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Synergy of high salt diet and infection with the sydney strain of Helicobacter pylori infection results in gastric mucosal dysplasia in mongolian gerbils
can be regarded as a nnvel marker in the epithelium associated with lesions preceeding gastric cancer. CD133+ stem cells originating from the bone marrow seem to be revolved in the reconstitntion if the gastric epithelium following destruction by li. pyiori.
overnight incubation with 5% C Q at 37~ vacuolated and hummingbird cells were counted directly using phase contrast microscopy. Statistical analysis was by' ANOVA and Student's paired t-test. Results: Co-culture with H. pylori strain 60190, but not its VaeA mutant, increased AGS cell vacuolation from 0.8 +_0.8 to 25,4 _+6.5%. Indomethacin had no significant effect in the absence of H. pylori, but inhibited H. pylori-induced vacuolation in a dosedependent manner (1001aM: by 56 + I4%, p = 0.02; ltxM: by 42 +_ 15 %, p = 0.05; 0.01}xM: by 26+_ 13%, p=0.26). Co-culture with H. pylori strain 60190, but not its CagE mutant increased AGS hummingbirds from 0 to 6.2 _+1.7%. Indomethacin had no effect in the absence of H. pylorL In the presence of H. pylori, indomethacin at 100~M surpnsingly increased hummingbirds by 240+_98%, p=0.05. Conclusion: lndomethacin reduces H. pflorMnduced vaeuohtion, but in contrast, at high concentrations, increases H. pyloriinduced cytoskeletal changes. We speculate that these actions contribute to NSAID4nduced reduction in H. pylori-associated gastric cancer risk.
W923
Alterations in Gastric Mucous Cell Kinetics, Part of the Defense System Against H. pylori Infection in the Regenerating Zone? Akifmin Tanaka, Hiruyoshi Ota, Tsntomu Katsuyama, Masayoshi Hayama, Kazuhiro Watanabe, Hitoshi lshida, Shin'iehi Takahashi Background: Gastric mucosa has two types of mucous cells: surface mucous cells (SMCs) and gland mucous cells (GMCs). Hdicobacter pylori (li. pyIori) do not attach to GMCs and mucins from GMCs inhibit the movement of li, pylori in the gastric mucous gel layer. H. pylon increase the cell kinetics ot gastric epithelial cells. AIM: To examine the effect of li. IEvlori intection on gastric epithelial cell kinetics from the viewpoint of the phenotype of gastric mucous ceils. Methods: Gastric mucosal biopsy specimens obtained from 10 nonintectad volunteers and 20 H, pylori infected patients before and after eradication were used. Formalin-fixed and paraffin-embedded tissue sections were examined by a sequential staining consisting ot immunoalkaline phosphatase method for Ki67, immunopemxidase method with monoclonal antibody HIK1083, and histochemical staining for SMCs (periodic acid oxidation-thionine Schiff reaction: PA-TS), Ki-67 labeling indices (Lls) and phenotype of proliferating gastric mucous cells were analyzed. Results: SMCs, GMCs and proliferating cells were simultaneously visualized in histological specimens; SMCs were stained blue with PA-TS, GMCs were stained red with liIK1083, and nuclei of proliferating cells stained brown with Ki-67. Proliferating SMCs were distributed in lower part of gastric foveola and proliferating GMCs were localized in the lower part ot gastric lm,eola around the generating zone. Ki-67 LIs were higher in H. pylori infected patmnts (0.15 in the corpus and 0,21 in the antrum) than in vohinteers (0.06 and 0.11) (p<0.001). Ratios of Ki-67 LIs of GMC to those of SMC (GMC/SMC ratio) were higher in H. pylori infected patients (0.18 in the corpus and 0.26 in the antrum) than in volunteers (0.08 and 0.08) (p<0.05). After the eradicanon of H. pylonS, the increased Ki-67 Lls and the GMC/SMC ratio in gastric mucosa of H. pyloriintected patterns decreased to ahnost normal levels. Conclusion: H. pylori increased cell kinetics of gastric mucous cells and increased the GMC/SMC ratio. The increased number of GMCs in the gastric generating zone may act as part of the defense system against H. pylori intectinn in the generating zone
W926
Helicobacter Heilmannii like Organism Invading into Parietal Cell Brought About Apoptosis Under Acid Suppressant Administration : Its Relation to Gastric Mucosal Atrophy Kaori Nishikawa, Masahiko Nakamnra, Hidenori Matsui, liitoshi Ishida, Shin'ichi Takahashi During our previous investigation of the mice gastric mucosa infected with Helicobacter heilmannii like organism (HIILO), these bacilli were found to be localized in the deeper fundic and amral glands, compared with lielicobacter pylori (llp) and HIILO invaded about 10% of the parietal cell into the intracelhilar canalicufi and some of these cells became necrotic. Thus the present study was undertaken to clarify whether the addition of acid suppressants to these HHLO infected mice could bring about more deleterious influence on the gastric epithelial ceils or not. Materials and methods: C3li mice infected with HIILO for 6 months and Mongolian gerbils infected with Hp for 6 months were used in the following experiments. After treatment with cimetidine (100mg/b.w.) or lansoprazole (30rag/ kg b.w.,every 12 hours for 3 days) through orogastric incubation, apoptotic fundic and antral glandular cells were detected by the Flow cytometry nsed Propidinm iodide. The localization of IIHLO and stem cell was concomitantly observed by the immunohistochemistry and electron microscopic cytochemistry using polyclonal antibodies against Helicobacter pyrori and Musashi-1, one of the RNA-binding protein found in stem cells. Results: The number of the apoptotic cells,mostly coinciding with parietal cells, signfl'icantly increased in the cimetidine treated group, but slightly in the lansoprazole-administered group. In addition, electron microscopic observation has clarified that HHLO was detected not only in the intracellular canaliculi but also in the cytoplasm of the parietal cells. In addition, some of the Musashi-l-posotive stem cells wrer directly attached by HHLO. On the other hand, few cells fell into apoptosis in the Hp-infected Mongolian gerbil fundic and antral mucosa and few Hp were detected near the stem cells. In conclusion, HHLO and not Hp was shown to bnng about the apoptosis of the parietal cells under the influence of H2 receptor antagonists but not by proton pump inhibitors. This observation suggests the necessity of eradication of HHLO before the long term treatment using acid suppressants to prevent the destruction of the parietal cells and induction of the gastric mucosal atrophy.
W924 Synergy of High Salt Diet and Infection with The Sydney Strain of Helicobacter Pylar/Infection Results in Gastric Mucosal Dysplasia in Mongolian Gerbils Mohamed Sagar, Ireneusz Padol, Catherine Streutker, Richard H. Hunt Background and aim: A high salt diet in humans and experimental animals is known to cause gastritis, and is considered as a gastnc cancer promoter. The animal experiments using the Mongolian gerbil have shown that long-term Helicobacter pylor~ (HP) infection induced gastric cancer We, therefore, designed a pilot study to determine whether excessive intake of dietary salt would have an effect on long term HP-infection and corresponding pathological changes in the stomach of Mongolian gerbils, Method: The gerbils were placed on either a normal or high salt (8%) diet. Pour weeks later the animals in both groups were once orally intected vath 1X108CFU of liP-Sydney strain (IIP-SS1). The gerbils were sacrificed at 62 weeks post infection (p.i). A sample of gastric mucosa was used for a rapid urease test, while the remaining stmnach was fixed in 10% formalin and stained with either H&E or Warthin-Starry stains. "['he presence of neutruphils, lymphocytes, plasmacytes, epithelial reaction, epithelial erosion, mucin depletion, atrophy', dysplasia/metaplasia, and bacterial density' were assessed histologically on a scale of 0-3. Results: Gerbils tolerated the high salt diet well during the whole period of the study. At 62 weeks after inoculation, colomzation with HP-SS1 was maintained in the stomach as confirmed by both rapid urease test and hstology, We have previously reported that the distribution of HP-SSI in animals on a high salt diet was patchy" and that atrophy was seen in this group of animals as early as 12 weeks [Gastroenterology 2002; 122: A 423]. At 62 weeks post mfection we observed changes suggestive of low-grade dysplasia only' in the gastric mucosa of gerbils fed a high salt diet. Also, inflammatmn scores were significantly higher in this group as compared with the infected gerbils on a normal diet. No gastric tumours were observed in a W of the expenmental groups, possibly because the number of animals was low Conclnsion: This study confirms that long-term intection with HP-SSI strain of Helicobacter pylori combined with a high salt diet increases inflammation resulting in higher inflammation scores and in dysplasia. Further studies using larger numbers of anunals are needed to validate a carcinogenic role of Hehcobactet pylori intKtion in this model.
w927
Fas Antigen ligation Signal for Apoptosis or Proliferation in Gastric Mucosal Cells - The Importance of Receptor Abundance liancben Li, Colin Stoicov, Jeanmarie Houghton Background&Aim lielicobacter pylon infection induces disease through immune mediated dysregulation of gastric mucosal apoptosis and proliteration. Fas pathway disruption dramatically decreases gastric mncosal apoptosis, proliferation and attenuates disease progression in Helicobacter-infected mice. In lymphocytes, Fas Ag figation has shown paradoxical signahng towards proliferation or apoptosis, dependent upon cellular and environmental factors; setting a precedent tbr differential Fas signaling. Key components of tile Fas Ag signaling pathway are differential up-regulated on diverse gastric cell populations during infection. Cells with the highest receptor abundance appear most susceptible to apoptosis, while cells within the proliferative zones expressing substantially" less receptor, are less susceptible to apoptosis. Therefore, the aim of this study' was to evaluate the relationship between surface Fas receptor abundance and growth outcomes of gastric mucosal cells. Methods. Rat gastric mucosal cells (RGM-1) were stably transfected with the pMSCW-puru retroviral vector containing the full ]enNh mouse Fas Ag cDNA. Low, moderate and high expressing clones were isolated and receptor abundance verified (RT-PCR, FACS analysis). Growth respomse to FasL was assessed by WST-1 assay, BrdU incorporation, NF-KB Luciferase Assay, Annexin V staining LLnL was used as an NF-tCB inhibitor. Results. Basal grm~r characteristics did not differ between clones. Exposure to 12.5-25ng/ml FasL (LD50 = 50ng/ml) was associated with >50% apoptosis in high expressing clones, and -< 4% in the RGM1/pMSCV-puro and low Fas Ag expressmg clones. Proliferation (BrdU incorporation) was increased 7.2_+ 1% and 5+_2% over control levels in low and moderate expressing clones, and consistently decreased (6+_2%) in high expression clones. NF-/CB activation was seen in all clones in response to ligand addition. Inhibition of NF-KB resulted in an increase in apoptosis in Fas expressing clones (but not control cells) and a decrease in proliferation. Conclusions. Fas receptor ligation in gastric epithelial cells resuhs in either apoptosis or proliferation depending on Fas receptor abundance. NF-KB activation appears to protect against Fas mediated apoptosis and contribute to proliferation.
W925
Indomethacin Alters Helicobacter pylori-Induced Effects on Gastric Epithelial Cells In Vitro Martin W. James, Ricbard H. Argent, Rachael ,J. Thonr~as, Christopher j. Hawkey, John C. Atherton hltruduction: Helicobacterpylori infection is a major risk tactor for distal gastric adenoearcinoma. Pathogenicity determinants include production of an active form of vacuolating cytotoxm (VacA) and possession of the cag pathogenicity island (cag Pal). Toxigenic strains cause marked epithelial cell vacuolation in vitro whilst cag-dependent effects include rearrangement of the actin cytoskeleton to form a "hummingbird" phenotype. NSMD ingestion is ~ i a t e d with a reduced incidence of gastric adenocarcinoma, although mechanisms are unclear. We tested the hypothesis that NSMDs modulate H. pylori-mduced effects on epithelial cells, in particular eftects induced by VacA and the tag Pal. Methods: AGS cells (1-2 x 105 cells/ml) were co-cultured with H. pylon strain 60190 (tag+, vacA sl/ml) or its VacA or CagE isogenic mutants at a bacteria:cell ratio of 0.02-70:1. Iodomethacin (a nonselective NSAID) or vehicle control was added at a concentration of 0.01-100taM. After
A-595
AGA
Abstracts
overnight incubation with 5% C Q at 37~ vacuolated and hummingbird cells were counted directly using phase contrast microscopy. Statistical analysis was by' ANOVA and Student's paired t-test. Results: Co-culture with H. pylori strain 60190, but not its VaeA mutant, increased AGS cell vacuolation from 0.8 +_0.8 to 25,4 _+6.5%. Indomethacin had no significant effect in the absence of H. pylori, but inhibited H. pylori-induced vacuolation in a dosedependent manner (1001aM: by 56 + I4%, p = 0.02; ltxM: by 42 +_ 15 %, p = 0.05; 0.01}xM: by 26+_ 13%, p=0.26). Co-culture with H. pylori strain 60190, but not its CagE mutant increased AGS hummingbirds from 0 to 6.2 _+1.7%. Indomethacin had no effect in the absence of H. pylorL In the presence of H. pylori, indomethacin at 100~M surpnsingly increased hummingbirds by 240+_98%, p=0.05. Conclusion: lndomethacin reduces H. pflorMnduced vaeuohtion, but in contrast, at high concentrations, increases H. pyloriinduced cytoskeletal changes. We speculate that these actions contribute to NSAID4nduced reduction in H. pylori-associated gastric cancer risk.
W923
Alterations in Gastric Mucous Cell Kinetics, Part of the Defense System Against H. pylori Infection in the Regenerating Zone? Akifmin Tanaka, Hiruyoshi Ota, Tsntomu Katsuyama, Masayoshi Hayama, Kazuhiro Watanabe, Hitoshi lshida, Shin'iehi Takahashi Background: Gastric mucosa has two types of mucous cells: surface mucous cells (SMCs) and gland mucous cells (GMCs). Hdicobacter pylori (li. pyIori) do not attach to GMCs and mucins from GMCs inhibit the movement of li, pylori in the gastric mucous gel layer. H. pylon increase the cell kinetics ot gastric epithelial cells. AIM: To examine the effect of li. IEvlori intection on gastric epithelial cell kinetics from the viewpoint of the phenotype of gastric mucous ceils. Methods: Gastric mucosal biopsy specimens obtained from 10 nonintectad volunteers and 20 H, pylori infected patients before and after eradication were used. Formalin-fixed and paraffin-embedded tissue sections were examined by a sequential staining consisting ot immunoalkaline phosphatase method for Ki67, immunopemxidase method with monoclonal antibody HIK1083, and histochemical staining for SMCs (periodic acid oxidation-thionine Schiff reaction: PA-TS), Ki-67 labeling indices (Lls) and phenotype of proliferating gastric mucous cells were analyzed. Results: SMCs, GMCs and proliferating cells were simultaneously visualized in histological specimens; SMCs were stained blue with PA-TS, GMCs were stained red with liIK1083, and nuclei of proliferating cells stained brown with Ki-67. Proliferating SMCs were distributed in lower part of gastric foveola and proliferating GMCs were localized in the lower part ot gastric lm,eola around the generating zone. Ki-67 LIs were higher in H. pylori infected patmnts (0.15 in the corpus and 0,21 in the antrum) than in vohinteers (0.06 and 0.11) (p<0.001). Ratios of Ki-67 LIs of GMC to those of SMC (GMC/SMC ratio) were higher in H. pylori infected patients (0.18 in the corpus and 0.26 in the antrum) than in volunteers (0.08 and 0.08) (p<0.05). After the eradicanon of H. pylonS, the increased Ki-67 Lls and the GMC/SMC ratio in gastric mucosa of H. pyloriintected patterns decreased to ahnost normal levels. Conclusion: H. pylori increased cell kinetics of gastric mucous cells and increased the GMC/SMC ratio. The increased number of GMCs in the gastric generating zone may act as part of the defense system against H. pylori intectinn in the generating zone
W926
Helicobacter Heilmannii like Organism Invading into Parietal Cell Brought About Apoptosis Under Acid Suppressant Administration : Its Relation to Gastric Mucosal Atrophy Kaori Nishikawa, Masahiko Nakamnra, Hidenori Matsui, liitoshi Ishida, Shin'ichi Takahashi During our previous investigation of the mice gastric mucosa infected with Helicobacter heilmannii like organism (HIILO), these bacilli were found to be localized in the deeper fundic and amral glands, compared with lielicobacter pylori (llp) and HIILO invaded about 10% of the parietal cell into the intracelhilar canalicufi and some of these cells became necrotic. Thus the present study was undertaken to clarify whether the addition of acid suppressants to these HHLO infected mice could bring about more deleterious influence on the gastric epithelial ceils or not. Materials and methods: C3li mice infected with HIILO for 6 months and Mongolian gerbils infected with Hp for 6 months were used in the following experiments. After treatment with cimetidine (100mg/b.w.) or lansoprazole (30rag/ kg b.w.,every 12 hours for 3 days) through orogastric incubation, apoptotic fundic and antral glandular cells were detected by the Flow cytometry nsed Propidinm iodide. The localization of IIHLO and stem cell was concomitantly observed by the immunohistochemistry and electron microscopic cytochemistry using polyclonal antibodies against Helicobacter pyrori and Musashi-1, one of the RNA-binding protein found in stem cells. Results: The number of the apoptotic cells,mostly coinciding with parietal cells, signfl'icantly increased in the cimetidine treated group, but slightly in the lansoprazole-administered group. In addition, electron microscopic observation has clarified that HHLO was detected not only in the intracellular canaliculi but also in the cytoplasm of the parietal cells. In addition, some of the Musashi-l-posotive stem cells wrer directly attached by HHLO. On the other hand, few cells fell into apoptosis in the Hp-infected Mongolian gerbil fundic and antral mucosa and few Hp were detected near the stem cells. In conclusion, HHLO and not Hp was shown to bnng about the apoptosis of the parietal cells under the influence of H2 receptor antagonists but not by proton pump inhibitors. This observation suggests the necessity of eradication of HHLO before the long term treatment using acid suppressants to prevent the destruction of the parietal cells and induction of the gastric mucosal atrophy.
W924 Synergy of High Salt Diet and Infection with The Sydney Strain of Helicobacter Pylar/Infection Results in Gastric Mucosal Dysplasia in Mongolian Gerbils Mohamed Sagar, Ireneusz Padol, Catherine Streutker, Richard H. Hunt Background and aim: A high salt diet in humans and experimental animals is known to cause gastritis, and is considered as a gastnc cancer promoter. The animal experiments using the Mongolian gerbil have shown that long-term Helicobacter pylor~ (HP) infection induced gastric cancer We, therefore, designed a pilot study to determine whether excessive intake of dietary salt would have an effect on long term HP-infection and corresponding pathological changes in the stomach of Mongolian gerbils, Method: The gerbils were placed on either a normal or high salt (8%) diet. Pour weeks later the animals in both groups were once orally intected vath 1X108CFU of liP-Sydney strain (IIP-SS1). The gerbils were sacrificed at 62 weeks post infection (p.i). A sample of gastric mucosa was used for a rapid urease test, while the remaining stmnach was fixed in 10% formalin and stained with either H&E or Warthin-Starry stains. "['he presence of neutruphils, lymphocytes, plasmacytes, epithelial reaction, epithelial erosion, mucin depletion, atrophy', dysplasia/metaplasia, and bacterial density' were assessed histologically on a scale of 0-3. Results: Gerbils tolerated the high salt diet well during the whole period of the study. At 62 weeks after inoculation, colomzation with HP-SS1 was maintained in the stomach as confirmed by both rapid urease test and hstology, We have previously reported that the distribution of HP-SSI in animals on a high salt diet was patchy" and that atrophy was seen in this group of animals as early as 12 weeks [Gastroenterology 2002; 122: A 423]. At 62 weeks post mfection we observed changes suggestive of low-grade dysplasia only' in the gastric mucosa of gerbils fed a high salt diet. Also, inflammatmn scores were significantly higher in this group as compared with the infected gerbils on a normal diet. No gastric tumours were observed in a W of the expenmental groups, possibly because the number of animals was low Conclnsion: This study confirms that long-term intection with HP-SSI strain of Helicobacter pylori combined with a high salt diet increases inflammation resulting in higher inflammation scores and in dysplasia. Further studies using larger numbers of anunals are needed to validate a carcinogenic role of Hehcobactet pylori intKtion in this model.
w927
Fas Antigen ligation Signal for Apoptosis or Proliferation in Gastric Mucosal Cells - The Importance of Receptor Abundance liancben Li, Colin Stoicov, Jeanmarie Houghton Background&Aim lielicobacter pylon infection induces disease through immune mediated dysregulation of gastric mucosal apoptosis and proliteration. Fas pathway disruption dramatically decreases gastric mncosal apoptosis, proliferation and attenuates disease progression in Helicobacter-infected mice. In lymphocytes, Fas Ag figation has shown paradoxical signahng towards proliferation or apoptosis, dependent upon cellular and environmental factors; setting a precedent tbr differential Fas signaling. Key components of tile Fas Ag signaling pathway are differential up-regulated on diverse gastric cell populations during infection. Cells with the highest receptor abundance appear most susceptible to apoptosis, while cells within the proliferative zones expressing substantially" less receptor, are less susceptible to apoptosis. Therefore, the aim of this study' was to evaluate the relationship between surface Fas receptor abundance and growth outcomes of gastric mucosal cells. Methods. Rat gastric mucosal cells (RGM-1) were stably transfected with the pMSCW-puru retroviral vector containing the full ]enNh mouse Fas Ag cDNA. Low, moderate and high expressing clones were isolated and receptor abundance verified (RT-PCR, FACS analysis). Growth respomse to FasL was assessed by WST-1 assay, BrdU incorporation, NF-KB Luciferase Assay, Annexin V staining LLnL was used as an NF-tCB inhibitor. Results. Basal grm~r characteristics did not differ between clones. Exposure to 12.5-25ng/ml FasL (LD50 = 50ng/ml) was associated with >50% apoptosis in high expressing clones, and -< 4% in the RGM1/pMSCV-puro and low Fas Ag expressmg clones. Proliferation (BrdU incorporation) was increased 7.2_+ 1% and 5+_2% over control levels in low and moderate expressing clones, and consistently decreased (6+_2%) in high expression clones. NF-/CB activation was seen in all clones in response to ligand addition. Inhibition of NF-KB resulted in an increase in apoptosis in Fas expressing clones (but not control cells) and a decrease in proliferation. Conclusions. Fas receptor ligation in gastric epithelial cells resuhs in either apoptosis or proliferation depending on Fas receptor abundance. NF-KB activation appears to protect against Fas mediated apoptosis and contribute to proliferation.
W925
Indomethacin Alters Helicobacter pylori-Induced Effects on Gastric Epithelial Cells In Vitro Martin W. James, Ricbard H. Argent, Rachael ,J. Thonr~as, Christopher j. Hawkey, John C. Atherton hltruduction: Helicobacterpylori infection is a major risk tactor for distal gastric adenoearcinoma. Pathogenicity determinants include production of an active form of vacuolating cytotoxm (VacA) and possession of the cag pathogenicity island (cag Pal). Toxigenic strains cause marked epithelial cell vacuolation in vitro whilst cag-dependent effects include rearrangement of the actin cytoskeleton to form a "hummingbird" phenotype. NSMD ingestion is ~ i a t e d with a reduced incidence of gastric adenocarcinoma, although mechanisms are unclear. We tested the hypothesis that NSMDs modulate H. pylori-mduced effects on epithelial cells, in particular eftects induced by VacA and the tag Pal. Methods: AGS cells (1-2 x 105 cells/ml) were co-cultured with H. pylon strain 60190 (tag+, vacA sl/ml) or its VacA or CagE isogenic mutants at a bacteria:cell ratio of 0.02-70:1. Iodomethacin (a nonselective NSAID) or vehicle control was added at a concentration of 0.01-100taM. After
A-595
AGA
Abstracts